RNA-seq screening of cuticle protein genes in Culex pipiens pallens among cypermethrin-resistant populations

Background A long-lasting overdependence on insecticides has led to the rapid spread of pyrethroid resistance in mosquito vectors, which poses a great risk to the general public. Although there are many studies on metabolic resistance and target resistance, few have investigated cuticle resistance and behaviour resistance. The cuticle of mosquitoes has been hypothesized to play a role in insecticide resistance by reducing penetration or sequestering insecticides.Methods We used RNA sequencing (RNA-seq) to analyse the transcriptome of cypermethrin-resistant and cypermethrin-susceptible strains of Culex pipiens pallens. We sequenced 6 samples using an Illumina HiSeq platform and generated approximately 6.66 Gb bases from each sample on average. Mapping the sequenced reads to a reference genome and reconstructing the transcripts via gene expression analysis, we detected differentially expressed genes (DEGs) among the samples. Followed Gene Ontology (GO) classification and functional enrichment. Finally, we screened the genes of cuticle proteins associated with drug resistance throughout the genome, selected the significant DEGs with a log2 fold change > 3.0 and Padj < 0.05, and applied real-time fluorescence quantitative polymerase chain reaction (PCR) to verify the DEGs. Results We obtained 13,517 novel transcripts, of which 8,653 were previously unknown splicing events for known genes, 665 were novel coding transcripts without any known features, and 4,199 were long non-coding RNA (lncRNA). A total of 1035, 944, and 657 genes were upregulated in comparisons between the samples, and 2680, 1215, and 975 genes were downregulated in comparisons between the samples. Finally, among all samples, 167 genes were upregulated and 145 genes were downregulated. The GO classification and functional enrichment of DEGs were as follows: molecular function, 224 genes; cellular component, 149 genes; and biological process, 272 genes. The expression of XM_001863852 and XM_001845881 in resistant strains of Culex pipiens pallens was lower than that in the laboratory sensitive strain, with 0.177 and 0.548-fold change in expression, respectively; the expression of the XM_001845883.1 in the resistant strain was higher than that in the susceptible strain, with a 2.281-fold change in expression.Conclusions Our results provide a reference for resistance mechanisms via the mosquito cuticle as well as a new perspective for disease vector control.